Resistance in the plant, Barbarea vulgaris, and counter-adaptations in flea beetles mediated by saponins.

Three saponins and two sapogenins had differential effects on food consumption in five near-isogenic flea beetle lines, which differ in their ability to utilize a novel host plant, Barbarea vulgaris (Brassicaceae). The ability to live on this plant is controlled by major, dominant R-genes in the flea beetle, Phyllotreta nemorum (Coleoptera: Chrysomelidae: Alticinae). A susceptible genotype (rr) is unable to live on the plant, whereas resistant genotypes (RR and Rr) can utilize the novel host plant. Among compounds isolated from B. vulgaris, hederagenin cellobioside (hederagenin-3-O-(4-O-beta-D-glucopyranosyl)-beta-D-glucopyranoside) inhibited feeding, whereas the effect of oleanolic acid cellobioside was much weaker. The aglycones (sapogenins) were inactive. Although hederagenin cellobioside was active against all flea beetle lines, its effect on food consumption was much stronger on the susceptible genotype (rr) compared to the resistant genotype (Rr). Susceptible and resistant flea beetle genotypes were equally sensitive to a non-host saponin, alpha-hederin (hederagenin-3-O-(2-O-alpha-L-rhamnopyranosyl)-alpha-L-arabinopyranoside). These results suggest that R-alleles in flea beetles might be specific adaptations to defensive saponins in B. vulgaris. A possible mechanism of action of the R-alleles might be to encode for an enzyme (e.g. a glucosidase), which is able to cleave glycosidic bonds in hederagenin cellobioside, but not in alpha-hederin. The potential role of saponins as defensive compounds in B. vulgaris and as targets for counter-adaptations in flea beetles and other insects is discussed.

Antiproliferative constituents from umbelliferae plants. IX. New triterpenoid glycosides from the fruits of Bupleurum rotundifolium.

The MeOH extract of the fruits of Bupleurum rotundifolium showed inhibitory activity against human gastric adenocarcinoma (MK-1) cell growth. Bioactivity-guided fractionation of the MeOH extract led to the isolation of four new triglycosides of 13beta,28-epoxy oleanane-type triterpenes, named rotundiosides O, Q, S and T; 12 new glycosides of oleanane-type triterpenes, named rotundiosides J-N, P, R, U-Y, and others; echinocystic acid 3-O-sulfate; and three known oleanane-type triterpene glycosides, rotundiosides A, F and G. The structures of the new isolates were determined based on chemical and spectroscopic evidence. The GI(50) of isolates against MK-1, HeLa and B16F10 cell lines are reported.

Antiproliferative constituents from Umbelliferae plants VII. Active triterpenes and rosmarinic acid from Centella asiatica.

The antiproliferative constituents in the MeOH extract from the aerial parts of Centella asiatica were investigated. Activity-guided fractionation of MeOH extract resulted in the isolation of ursolic acid lactone, ursolic acid, pomolic acid, 2alpha,3alpha-dihydroxyurs-12-en-28-oic acid, 3-epimaslinic acid, asiatic acid, corosolic acid, and rosmarinic acid. Antiproliferative activity of the isolated compounds against human gastric adenocarcinoma (MK-1), human uterine carcinoma (HeLa), and murine melanoma (B16F10) cells was estimated.

Interactions of phytoestrogens with estrogen receptors alpha and beta (III). Estrogenic activities of soy isoflavone aglycones and their metabolites isolated from human urine.

Two glucuronides (4'-O-, and 7-O-) and a glucuronyl (7-O-) sulfate (4'-O-) of genistein, two glucuronides (4'-O-, and 7-O-) and a glucuronyl (7-O-) sulfate (4'-O-) of daidzein, 7-O-glucuronides of glycitein, dihydrodaidzein and O-desmethylangolensin were isolated from the urine of volunteer subjects fed soy bean curds (Tofu). The estrogenic activities, i.e., i) the effect on the estrogen-dependent growth of MCF-7 cells, ii) the binding ability to human estrogen receptors (hERs) alpha and beta, and iii) the effect on hER-dependent beta-galactosidase induction, of these isoflavone metabolites were examined. Two synthetic isoflavone aglycones (dihydrodaidzein and O-desmethylangolensin) and four synthetic sulfates (4'-O- and 4'-, 7-di-O-) of genistein and daidzein were also studied for their estrogenic activities for the purpose of comparison. With respect to estrogenic acivity, the tested isoflavone metabolites were classified into three groups. The first group shows a very poor stimulatory effect toward the growth of MCF-7 cells, binding activity, and beta-galactosidase induction. The sulfates belong to this group. The second group shows a moderate binding activity but poor stimulation and beta-galactosidase induction. Some glucuronyl conjugates belong to this group. The last group shows a moderate stimulation and beta-galactosidase induction but poor binding activity. A mixed type of conjugates having glucuronyl and sulfony moieties belong to this group.

Antiproliferative activity of the main constituents from Phyllanthus emblica.

Eighteen main compounds, including four norsesquiterpenoids (1-4) and 14 phenolic compounds (5-18) isolated previously from Phyllanthus emblica, together with a main constituent, proanthocyanidin polymers (19) identified at this time from the roots, were estimated for their antiproliferative activities against MK-1 (human gastric adenocarcinoma), HeLa (human uterine carcinoma), and B16F10 (murine melanoma) cells using an MTT method. All of the phenolic compounds including the major components 5-8 from the fruit juice, 8, 9, and 12 from the branches and leaves, and 19 from the roots showed stronger inhibition against B16F10 cell growth than against HeLa and MK-1 cell growth. Norsesquiterpenoid glycosides 3 and 4 from the roots exhibited significant antiproliferative activities, although their aglycon 1 and monoglucoside 2 showed no inhibitory activity against these tumor cells.

Hepatoprotective constituents in plants. 14. Effects of soyasapogenol B, sophoradiol, and their glucuronides on the cytotoxicity of tert-butyl hydroperoxide to HepG2 cells.

The effects of soyasapogenol B, sophoradiol, their glucuronides, and glycyrrhizin on the hepatotoxicity of tert-butyl hydroperoxide (t-BuOOH) in a human-liver-derived cell line (HepG2 cells) were investigated. Glycyrrhizin showed significant dose-dependent protective effects against the cytotoxicity of t-BuOOH. Among soyasapogenol B and its glucuronides, the monoglucuronide showed the most potent hepatoprotective activity, followed by soyasapogenol B itself. Soyasaponin III was weakly protective, while soyasaponin I increased the toxicity of t-BuOOH. Among sophoradiol and its glucuronides, sophoradiol itself showed the most potent hepatoprotective activity, which was equal to glycyrrhizin, while the monoglucuronide and kaikasaponin III showed an increase in cytotoxicity. These results were considerably different from those reported previously on the protective effects of these compounds using primary cultures of immunologically injured rat liver cells. Consequently, the hepatoprotective action of the triterpene derivatives investigated would be different in HepG2 cells and in rat primary hepatocyte cultures.

Antiproliferative constituents from Umbelliferae plants VI. New ursane-type saikosaponin analogs from the fruits of Bupleurum rotundifolium.

The MeOH extract of the fruits of Bupleurum rotundifolium showed inhibitory activity against human gastric adenocarcinoma (MK-1) cell growth (GI(50): 6.25 microg/ml). From this extract, 10 new ursane-type triterpene glycosides viz. three 3-O-triosides (called rotundifoliosides A, I, and J) of 13beta,28-epoxy-3beta,16alpha-dihydroxyurs-11-ene, two (G and H) of 13beta,28-epoxy-3beta,16alpha,23-trihydroxyurs-11-ene, two (E and F) of 13beta,28-epoxy-3beta,16alpha,21beta-trihydroxyurs-11-ene, two (B and C) of 3beta,11alpha,16alpha,28-tetrahydroxyurs-12-ene, and one (D) of 3beta,11alpha,28-trihydroxy-15alpha,16alpha-epoxyurs-12-ene were isolated in addition to 16 new oleanane-type triterpene glycosides (rotundiosides J-Y), echinocystic acid 3-O-sulfate and 3 known oleanane-type triterpene glycosides (rotundiosides A, F and G). The isolation, structures and antiproliferative activity of the new ursane-type triterpene glycosides against MK-1, human uterus carcinoma (HeLa), and murine melanoma (B16F10) cell lines are described with some comments on the structural requirements for their activity.

Ursolic acid as a trypanocidal constituent in rosemary.

The MeOH extract of the leaves of rosemary (Rosmarinus officinalis L.) completely inhibited the motility of cultured epimastigotes of Trypanosoma cruzi at the concentration of 2 mg/ml after 2 h of incubation. Activity-guided fractionation of the MeOH extract has resulted in the isolation of three triterpene acids, betulinic, oleanolic and ursolic acids. Ursolic acid stopped the movement of all T. cruzi epimastigotes at the minimum concentration (MC(100)) of 40 micro g/ml (88 micro M) after 48 h of incubation. Oleanolic acid was less active (MC(100): 250 micro g/ml, 550 micro M) and betulinic acid was practically inactive.

Antiproliferative sesquiterpene lactones from the roots of Inula helenium.

The MeOH extract of the roots of Inula helenium showed a high inhibitory activity for cell growth against MK-1, HeLa and B16F10 cell lines. Significant activity was found in the hexane-soluble fraction. From the hexane-soluble fraction, seven sesquiterpenes, namely, one germacrane (4beta,5alpha-epoxy-1(10),11(13)-germacradiene-8,12-olide), one elemane (igalane), and five eudesmanes (alantolactone, isoalantolactone, 11alpha,13-dihydroalantolactone, 11alpha,13-dihydro-isoalantolactone, 5-epoxyalantolactone) were isolated. In vitro antiproliferative activities of the isolates against MK-1, HeLa and B16F10 cells are reported.

Activity-guided fractionation of green tea extract with antiproliferative activity against human stomach cancer cells.

Epidemiological studies have suggested that the consumption of green tea provides protection against stomach cancer. Fractionation of green tea extract, guided by antiproliferative activity against human stomach cancer (MK-1) cells, has resulted in the isolation of six active flavan-3-ols, epicatechin (EC), epigallocatechin (EGC), epigallocatechin gallate (EGCg), gallocatechin (GC), epicatechin gallate (ECg), gallocatechin gallate (GCg), together with inactive glycosides of kaempferol and quercetin. Among the six active flavan-3-ols, EGCg and GCg showed the highest activity, EGC, GC, ECg followed next, and the activity of EC was lowest. These data suggest that the presence of the three adjacent hydroxyl groups (pyrogallol or galloyl group) in the molecule would be a key factor for enhancing the activity. Since reactive oxygen species play an important role in cell death induction, radical scavenging activity was evaluated using the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical. The order of scavenging activity was ECg > or = EGCg > or = EGC > or = GC > or = EC. The compounds having a galloyl moiety showed more potent activity. The contribution of the pyrogallol moiety in the B-ring to the scavenging activity seemed to be less than that of the galloyl moiety.

Antiproliferative constituents in plants 10. Flavones from the leaves of Lantana montevidensis Briq. and consideration of structure-activity relationship.

The flavonoid fraction from the leaves of Lantana montevidensis Briq. (Verbenaceae) showed antiproliferative activity against human gastric adenocarcinoma (MK-1, GI50: 12 microg/ml), human uterus carcinoma (HeLa, 5 microg/ml), and murine melanoma (B16F10, 5 microg/ml) cells in vitro. Bioactivity-guided chemical investigation of the fraction has resulted in the isolation of apigenin (10) and ten 5,6,7-oxygenated flavones: cirsilineol (1), eupatorin (2), 5,4'-dihydroxy-6,7,3',5'-tetramethoxyflavone (3), 5,6-dihydroxy-7,3',4'-trimethoxyflavone (4), 5,6,4'-trihydroxy-7,3',5'-trimethoxyflavone (5), 5,6,3'-trihydroxy-7,4'-dimethoxyflavone (6), 5,3',4'-trihydroxy-6,7,5'-trimethoxyflavone (7), cirsiliol (8), hispidulin (9), and eupafolin (11). Antiproliferative activity of the isolated flavones, some other related flavones (luteolin, baicalein, 6-hydroxyluteolin, pectolinarigenin, jaceosidin, desmethoxycentaureidin, eupatilin, and chrysin) from other plant materials, and synthetic 6- and 7-methoxyflavones was evaluated, and the structure-activity relationships were examined.

Antiproliferative constituents in plants 9. Aerial parts of Lippia dulcis and Lippia canescens.

The antiproliferative constituents in the MeOH extracts of the aerial parts of Lippia dulcis Trev. and Lippia canescens Kunth (Verbenaceae) were investigated. Activity-guided chemical investigation of the MeOH extracts resulted in the isolation of the three bisabolane-type sesquiterpenes [(+)-hernandulcin (1), (-)-epihernandulcin (2), and (+)-anymol (3)] and four phenylethanoid glycosides [acteoside (4), isoacteoside (5), martynoside (6), and a new diacetylmartynoside (7)] from the former, and four phenylethanoid glycosides [acteoside (4), isoacteoside (5), arenarioside (8), and leucosceptoside A (9)] and three flavones [desmethoxycentaureidin (10), eupafolin (11), and 6-hydroxyluteolin (12)] from the latter. Antiproliferative activity of the isolated compounds against murine melanoma (B16F10), human gastric adenocarcinoma (MK-1), and human uterine carcinoma (HeLa) cells was estimated. (+)-Anymol (3), acteoside (4), isoacteoside (5), arenarioside (8), eupafolin (11), and 6-hydroxyluteolin (12) had GI50 values of 10-16 microM against B16F10 cell. Desmethoxycentaureidin (10) and eupafolin (11) showed high inhibitory activity against HeLa cell growth (GI50 9 microM, and 6 microM, respectively).

Identification of a triterpenoid saponin from a crucifer, Barbarea vulgaris, as a feeding deterrent to the diamondback moth, Plutella xylostella.

Larvae of the diamondback moth, Plutella xylostella, a crucifer specialist, refuse to feed on a crucifer, Barbarea vulgaris, because of the presence of a feeding deterrent, which is extractable with chloroform. We isolated a feeding deterrent from B. vulgaris leaves, by successive fractionations with silica-gel, ODS, i.e., C18 reversed phase, and Sephadex LH-20 column chromatographies, and ODS-HPLC, guided by a bioassay for feeding deterrent activity. The structure of the compound was determined to be a monodesmosidic triterpenoid saponin, 3-O-[O-beta-D-glucopyranosyl-(1-->4)-beta-D-glucopyranosyl]-hederagenin, based on FAB-MS, 1H- and 13C-NMR spectra, and hydrolysis experiments. When the compound was applied to cabbage leaf disks at greater than 0.18 microg/mm2, consumption of the disks by third instars was less than 11% of control disks treated with the solvent alone. Furthermore, all first instars died on the disks treated with the same concentrations. Because the concentration of the compound in the fresh leaves of B. vulgaris was comparable to the effective dose in the cabbage leaf disk tested, we conclude that the unacceptability of B. vulgaris to P. xylostella larvae is primarily due to this saponin.